Glucose homeostasis is regulated by the orchestrated secretion of hormones, primarily insulin and glucagon, from the pancreatic islet of Langerhans. Although the mechanism of glucose-stimulated insulin secretion (GSIS) is well-established, the molecular mechanisms that underlie the modulation of insulin and glucagon secretion by G-protein coupled receptors (GPCRs) are not clearly understood. This is especially significant considering GPCR ligands (i.e. GLP-1 analogs) have emerged as a promising treatment of Type-2 diabetes. Neuropeptide-Y (NPY) is a ubiquitous peptide messenger that is locally produced in pancreatic islets and decreases GSIS through Gi-mediated inhibition of adenylyl cyclase and reduced cAMP. In contrast, NPY has been shown to stimulate glucagon secretion from mouse and rat islets. Somatostatin (SST) is secreted by the pancreatic / cells and has two active forms-SST-14 and SST-28. Both forms bind with high affinity to SSTR2 and SSTR5, which are expressed abundantly in islets. Like NPY, SST inhibits insulin secretion through the Gi pathway, but it is reported also to have an inhibitory effect on glucagon secretion. To understand the differential effects of these two GPCR ligands, we propose to measure the effects of NPY and SST signaling on islet cellular metabolism, electrical activity, and hormone secretion. Here, it is hypothesized that NPY inhibits insulin secretion downstream and stimulates glucagon secretion upstream of Ca2+ signaling. SST alters pathways both upstream and downstream of Ca2+ signaling, inhibiting insulin and glucagon secretion through a similar mechanism. Two specific aims are proposed: 1) Determine the mechanisms by which NPY and SST inhibit -cell insulin secretion;2) Determine the signaling pathways of glucagon secretion modulated by NPY and SST. The proposed experiments combine novel and conventional imaging and electrophysiology techniques to address the aims. The use of a transgenic mouse model permits definitive .-cell identification and enhances our ability to characterize islet signaling pathways. The cellular redox state will be measured by the combined autofluorescence signal from NADH and NADPH (NAD(P)H);this assay will be used to investigate the effects of NPY and SST on cellular metabolism. Activation of voltage-gated Ca2+ channels and subsequent membrane depolarization are necessary components of insulin and glucagon secretion, and these events can be measured using electrophysiological and fluorescence approaches. Insight into ion channel function, Ca2+ signaling, and exocytosis in the presence and absence of NPY or SST will help elucidate the mechanism(s) by which these inhibitory ligands modulate insulin and glucagon secretion. These biophysical techniques will be correlated with traditional hormone secretion measurements and biochemical assays. The resulting quantitative data will allow us to narrow down and, eventually, determine the precise molecular pathways modulated by these GPCR ligands. PUBLIC HEALTH RELEVANCE: To wholly understand the American epidemic that is Type-2 diabetes, we need to understand the complex regulation of hormone secretion from pancreatic islets. Changes in insulin and glucagon secretion may be a compensatory mechanism by which the body attempts to overcome insulin-resistance during Type-2 diabetes. Insight into the mechanisms by which insulin and glucagon secretion are optimized by other hormones and the nervous system will allow for a more comprehensive understanding of the pathology of Type-2 diabetes, and will thus contribute to the development of future therapies and the overall health of the nation.